POS0229 ELUCIDATING THE MECHANISM OF ACTION DOWNSTREAM OF ROR2 BLOCKADE: A PHOSPHOPROTEOMIC APPROACH
Authors: A. S. Thorup, J. Wilson, P. Cutillas, F. Dell’accio
Journal: Annals of the Rheumatic Diseases (2022)

Background Osteoarthritis (OA) affects 12% of the population, and yet we still have no disease-modifying treatment. Cartilage breakdown is the hallmark of OA, and patients suffer from pain and loss of joint function/independence, severely affecting quality of life. Therefore, there is a huge unmet clinical need. Receptor tyrosine kinase–like orphan receptor 2 (ROR2) is a non-canonical WNT receptor that regulates the planar cell polarity pathway, controlling limb outgrowth. During skeletal development chondrocytes require ROR2 to undergo hypertrophy in the process of endochondral bone formation (1). Loss of function mutations in humans causes Recessive Robinow Syndrome (limb shortening and brachydactyly (2,3)). Although absent from healthy adult articular cartilage, our initial studies identified high ROR2 expression in chondrocytes from patients with OA, suggesting a role in the disease process. In a murine model of OA, blocking ROR2 in therapeutic regime using siRNA resulted in reduced cartilage destruction and rapid and sustained pain relief. Due to the limited expression pattern of ROR2 in adulthood, no systemic or local toxicity were expected, nor were any observed. We also found that blocking ROR2 supports cartilage formation in a human cartilage organoid model in nude mice using chondrocytes from patients with OA, and proposed ROR2 blockade as a potential disease-modifying OA treatment. The mechanism of action of ROR2 blockade was independent of modulation of canonical WNT signaling. YAP inhibition was required, but not sufficient, for the chondrogenic effect (4). Therefore, additional, yet unknown mechanisms must be involved downstream of ROR2 blockade. Objectives To study the phosphorylation events downstream of ROR2, to further our understanding of the mechanism of action of ROR2 blockade. Methods Phosphoproteomics (label free liquid chromatography tandem-mass spectrometry with TiO2 based phospho-enrichment), in vitro studies in cells, CRISPR. Results WNT5A is the most well established ligand for ROR2. We confirmed that WNT5A stimulation leads to time-dependent phosphorylation of ERK in C3H10T1/2 cells. We then deleted ROR2 in C3H10T1/2 cells by CRISPR, and performed phosphoproteomics analysis on wildtype (control) C3H10T1/2 cells and ROR2-CRISPR C3H10T1/2 cells stimulated with 100ng/ml recombinant WNT5A for 5 or 15 minutes. ROR2-dependant phosphorylation targets were defined as proteins differentially phosphorylated in control cells upon WNT5A stimulation, but that did not occur in ROR2-CRISPR cells. YAP was phosphorylated by WNT5A in a ROR2-dependant manner. WNT5A/ROR2 also modulated the YAP signaling pathway at other levels. KEGG analysis revealed that other pathways with known roles in the pathogenesis of OA were enhanced, including NF-KB, mTOR signaling and cellular senescence. With the current technology, ROR2 blockade requires intra-articular injections of siRNA conjugated to atelocollagen every 5 days. Preliminary efficacy data of potentially longer-acting ROR2 blockers are promising. Conclusion ROR2 blockade has potential as a disease-modifying treatment for OA, resulting in cartilage protection and rapid and sustained pain relief in a murine model. Here, we studied the mechanism downstream of ROR2 blockade using a phosphoproteomics approach. We confirmed that YAP signaling was modulated by ROR2, and identified novel pathways and cellular processes regulated by WNT5A/ROR2. Further studies are needed to clarify their role downstream of ROR2 blockade in OA. Our current siRNA-atelocollagen based technology requires intra-articular injections too frequently to be acceptable for patients. We are developing ROR2 blockade which can be administered systemically or intra-articularly not more often than every 3 months. References [1]DeChiara, T. M. et al. Nat. Genet. 24 , 271–4 (2000). [2]Bokhoven, H. Van, Celli, J. &amp; Kayserili, H. Nature 25 , 423–426 (2000). [3]Afzal, A., Rajab, A., Fenske, C. &amp; Oldridge, M. Nature 25 , 419–422 (2000). [4]Thorup, A.-S. et al. Sci. Transl. Med. 12 , eaax3063 (2020). Disclosure of Interests Anne-Sophie Thorup: None declared, Jacob Wilson: None declared, Pedro Cutillas: None declared, Francesco Dell’Accio Consultant of: Samumed - not related to this abstract, Grant/research support from: UCB - not related to this abstract